1. Field of the Invention
This invention is related generally to the preservation of viable cells and in particular to a medium for conditioning and long term storage of viable cells.
2. Discussion of the Related Art
The preservation of viable cells which have been harvested from a donor source is of great importance and utility in the scientific and medical communities. Indeed, cells which have been harvested and preserved are routinely used in scientific research and development. For instance, preserved cells are often tested to aid in the development of medical treatments or to provide information on physical or chemical properties of the cells. Further, a collection of readily available viable cells allows scientists to conduct experiments at times which are suitable to laboratory availability or the researcher""s schedule. To be useful, the preserved cells must retain the integrity and viability of the cells at the time of harvest. Thus, the process of preserving the cells must not, in itself, damage or destroy the cells.
In conventional cryopreservation techniques, cells are harvested, suspended in a storage solution, then preserved by freezing. When the cells are to be used, they are thawed, for example, cells taken from human donor sources are brought back to the normal human body temperature (i.e., approximately 37xc2x0 C.), and then placed in a cell culture medium. Cryopreservation protocols subject the cells to a multitude of stresses and insults throughout the process of cell harvesting, freezing, and thawing. These stresses and insults can cause irreparable damage to the cell.
Ischemia, a lack of blood flow, occurs as soon as the life of the cell""s donor is terminated. Immediately thereafter, the cell experiences hypoxia, or oxygen deprivation, due to the lack of blood flow. Hypoxia causes anaerobic metabolism in normally aerobic cells. Anaerobic metabolism produces toxic byproducts, such as the build-up of lactic acid (acidosis). Some of the byproducts of anaerobic metabolism produce oxygen free-radicals that damage or destroy the cells when the cells are reoxygenated. Accordingly, prior to taking a tissue sample, the temperature of the donor source is reduced such that metabolic activity in the cells of the donor source is minimized. Reduction of temperature of the donor source reduces the energy state of the cells which aids in reducing the affects of ischemia and hypoxia. Typically, the temperature of the donor source is lowered to 4xc2x0 C. Although some residual metabolic activity exists in the cells at 4xc2x0 C., 4xc2x0 C. is about the lowest temperature available which does not cause the formation of ice crystals on the cells.
The cells are typically isolated from the tissue sample by addition of a hydrolytic enzyme. The hydrolytic enzyme deteriorates the extracellular tissue structure, thus causing the release of the desired cells. Unfortunately, the hydrolytic enzyme also harms the isolated cells. Some of the cells are destroyed during isolation. Other cells are weakened by exposure to the hydrolytic enzyme. It is believed that cells weakened by such enzymatic insult are less viable.
Once the cells are harvested, the cells are suspended in a storage solution which is also, typically, at 4xc2x0 C. One example of a widely used cell storage medium is Dulbecco""s Modified Eagle Medium (xe2x80x9cDMEMxe2x80x9d), an aqueous solution containing 10 wt. % fetal calf serum and low levels of glucose. DMEM is intended to provide support for the minimal metabolic activity which occurs just before the cells are frozen and just after the cells are brought to normal body temperature. Because cells are frozen as soon after harvest as possible, in order to completely arrest cell metabolism, the amount of glucose needed to support metabolic activity is quite low.
The harvested cells can also be harmed by the initial freezing and the subsequent thawing of the cell suspension. The cell membranes can be damaged primarily due to the rapid change in osmotic pressure that results when liquid inside or outside the cell is frozen or thawed. Freezing and thawing of the cell suspension causes a dramatic change in the concentration of liquid on one side of the membrane relative to the other. The dramatic change in concentration creates an osmotic pressure differential. The transmembrane pressure differential causes liquid to flow into the cell or liquid in the cell to flow out of the cell to reach equilibrium osmotic pressure. When excess liquid flows into a cell, the cells burst. When too much liquid leaves a cell, the cell shrivels and dies.
It is known to add cryopreservatives to a cell storage medium, such as DMEM, to prevent cell damage during freezing. Cryopreservatives include dimethyl sulfoxide (DMSO), glycerol, propylene glycol, and other large molecules with a high bonding affinity to water. Cryopreservatives are absorbed into the cells and have sufficient size that they are not likely to be rapidly transported across the membrane. Thus, when osmotic pressures change, the water remains bound to the cryopreservative and is stabilized to the change in transmembrane osmotic pressure. Of all the cryopreservatives, DMSO is by far the most preferred because of its high bonding affinity to water. However, DMSO is toxic to cells if added when the cells are at normal body temperature, and it is generally rapidly added to the cells just before the cells are frozen, i.e., when the temperature of the cells has been lowered to approximately 4xc2x0 C. Furthermore, the cells must be carefully washed to remove DMSO after the cells are subsequently thawed to a temperature of about 4xc2x0 C.
In addition to the above-stated problems, current preservation protocols are limited in that they are not necessarily transferrable between samples. Indeed, the type of sample has, in part, dictated the requirements of the preservation technique such that the technique employed is dependent, in part, upon the sample to be stored. Examples of various techniques of freezing and thawing of different sample types are found, for example, in U.S. Pat. No. 4,004,975 to Lionetti et al., directed to freezing and thawing of human white cells; U.S. Pat. No. 4,890,457 to McNally et al., directed to the freezing and thawing of collagen-rich tissue, such as heart valves; and U.S. Pat. No. 4,965,185 to Grischenko et al., directed to the freezing and thawing of embryos, more specifically, mammal embryos.
U.S. Pat. No. 5,328,821 to Fischer et al. discloses a cryopreservation solution for tissue slices. The solution contains (a) glucose and (b) a cryopreservative. Other ingredients include (c) impermeates, such as potassium gluconate, potassium saccharate, and mannitol, to prevent or minimize hypothermic induced cell swelling, (d) hydrogen ion buffers, such as a phosphate, (e) adenosine, an ATP precursor for the regeneration of high energy phosphate compounds, (f) free-radical inhibitors, such as allopurinol and mannitol, (g) reducing agents, such as glutathione, (h) inorganic salts, such as KCl, MgSO4, MgCl, NaHCO3, and KHCO3, (i) vitamins, such as vitamin E and vitamin C, (j) hormones, such as dexamethasone and insulin, (k) calcium channel blockers, such as verapamil, and (1) acid generating substrates, such as succinate, fructose and glucose. One of the drawbacks of the cell cryopreservation solution described in the Fischer et al. patent is that its use is limited to cryopreservation of tissue slices. Consequently, the utility of the solution disclosed in the Fischer patent has unproven effectiveness with harvested cells that have been weakened because they have been isolated by treatment with a hydrolytic enzyme.
Although some cryopreservation protocols have altered the conventional methods, these altered methods have failed to address all of the above mentioned problems. For instance, U.S. Pat. Nos. 5,171,660 and 5,424,207, both to Carpenter et al., describe an alternative to the immediate freezing of tissue samples. These patents give examples of placing heart leaflets in DMEM and then preincubating the tissue, for from about 5 minutes to about 24 hours, at a temperature of from about 27xc2x0 C. to about 42xc2x0 C. The preincubation is said to assure that the metabolic energy status and functional capacity of the tissue are restored when the tissue is thawed.
In U.S. Pat. No. 4,559,298 to Fahy, directed to vitrification of biological material, the cryopreservative is introduced and removed in step-wise concentrations. Specifically, the method in Fahy uses step-wise concentrations of greater than 10% per step. The large step-wise additions are aimed at inhibiting re-establishment of the isotonic volume of the cells prior to vitrification, i.e., osmotic equilibrium of the cells is not desired. Further, despite the step-wise addition and removal of a cryopreservative, intra-cellular concentrations are about 30% which is too high of a concentration and is not acceptable for some sample types, such as, for example, eukaryotes and aerobic prokaryotes. In U.S. Pat. No. 4,890,457 to McNally et al., which is directed toward collagen-rich tissue, the cryopreservative, DMSO, is removed in a 2.5% step-wise concentration. Nonetheless, the cryopreservative is not introduced in the same gradual step-wise concentrations, thus, potentially introducing cell stresses prior to freezing.
Thus, there remains a need for a generally applicable method for cell cryopreservation that more effectively maintains the integrity, viability and function of all types of cells during the cryopreservation process, more specifically, eukaryote, and aerobic prokaryote cells. The present invention satisfies these and other needs and provides further related advantages.
The present invention is a universally-adaptable cell preconditioning and storage medium that can be used for the effective cryopreservation of cells at temperatures less than 4xc2x0 C. The aqueous medium contains adenosine, a calcium channel blocker, and a nutrient-rich matrix that has a sufficient amount of cell metabolites to sustain the metabolic needs of the harvested cells while incubating the cells, generally at a temperature of from about 35xc2x0 C. to about 38xc2x0 C., preferably, about 37xc2x0 C. for a period of at least 10 minutes, preferably about 25 minutes, without producing detectable levels of lactate or substantially depleting the metabolic substrates of the cell.
Adenosine is included in the cell medium, typically in a concentration ranging from about 2.7 mM to about 3.6 mM. Preferably, the adenosine concentration ranges from about 2.9 mM to about 3.1 mM, and, more preferably, is about 3.0 mM. The adenosine is rapidly converted by the cells into adenosine triphosphate to supply immediate energy to the cells during preconditioning. The medium can also include other cell energy sources, such as saccharides like glucose, or metabolites of glucose, such as pyruvate.
A calcium channel blocker is included in the cell medium to prevent calcium-regulated membrane transport of the adenosine. An example of a preferred calcium channel blocker is verapamil. In one embodiment, verapamil is added to the cell medium in an amount ranging from about 0.04 mM to about 0.07 mM. It is added preferably in an amount ranging from about 0.05 mM to about 0.06 and more preferably in an amount of about 0.05 mM.
The other cell metabolites include nutrients that are easily absorbed into the cells to be preserved. Representative nutrients include one or more amino acids selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Additionally, the cell metabolites. preferably include one or more vitamins selected from the group comprising pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin, and thiamine. The concentration of the amino acids is chosen to match the amino acid concentration found in the healthy cytoplasm of the cells to be preconditioned. Alternatively, the concentration of amino acids in the medium is chosen to be proportional to the metabolic needs of the cells during normal cell metabolism.
In some embodiments, the cell medium includes an inorganic salt. Suitable salt-forming inorganic anions include chloride, phosphate, sulfate, and selenite. Suitable salt-forming inorganic cations include sodium, potassium, magnesium, copper, and zinc cations. And in some embodiments, the cell medium has a concentration of inorganic salts substantially equal to the concentration of inorganic salts found in the in vivo donor cells.
Also in some embodiments, the cell cryopreservation medium contains at least one hormone. Representative hormones include insulin, and preferably bovine pancreas insulin; dexamethasone; leutropic hormone and preferably sheep leutropic hormone; transferrin and preferably human transferrin; somatropin; linoleic acid; fetal bovine serum.
In some embodiments, the medium also contains a cryopreservative for protecting the cells during freezing and thawing. Preferably, the cryopreservative is DMSO.
It is also preferable for the medium to include a oxygen free radical scavenger to protect the cells from oxygen free radicals produced during reoxygenation after storage. Most preferably, the oxygen free radical scavengers are allopurinol and/or glutathione. Alternatively, the cell salvage medium comprises glycine, glutamine, glutamic acid, and cysteine, proteins that are rapidly converted by the cell into glutathione.
The cell medium preferably is buffered with a mild buffer solution having a content and concentration such that the cell medium has a first pH that ranges from about 7.3 to about 7.5 at a temperature above 35xc2x0 C. and has a second pH ranging from about 6.3 to about 6.6, preferably form about 6.4 to about 6.5, and most preferably about 6.4, at a temperature below about 4xc2x0 C. A suitable buffer includes a sodium carbonate buffer, an N-[Hydroxyethyl]piperazine-Nxe2x80x2[2-ethananesulfonic acid] (xe2x80x9cHEPESxe2x80x9d) buffer or a combination of the two. Overall, the concentration of all of the components preferably create a solution that is slightly hyperosmolar.
The present invention is also directed to a preserved cell sample, that includes a sample of eukaryotic or prokaryotic cells, suspended and frozen, preferably at the boiling point of liquid nitrogen, in the cell cryopreservation medium, as well as to a kit for preserving a sample of cells comprising a storage vessel and the cell cryopreservation and cryopreservation medium. In some embodiments, the kit further includes instructions to incubate cell at 37xc2x0 C. for a period ranging from about 10 minutes to about 1 hour and preferably about 25 minutes prior to storing the cells at a temperature below 4xc2x0 C. Preferably, the kit also includes an oxygen source for oxygenating the cell preconditioning and cryopreservation and medium during the preconditioning. Most preferably, the kit includes instructions to oxygenate the solution with a gas having an oxygen content of no less than 80 vol. % while the cells are incubated at 37xc2x0 C.
The present invention is a universally-adaptable, aqueous cell preconditioning and cryopreservation medium useful for preserving cells at temperatures less than 4xc2x0 C. and in some embodiments less than 0xc2x0 C. The same medium is used to first precondition the cells thereby effectively reinvigorating cells damaged by harvesting from a donor source. Unlike conventional cell storage media, the inventive cell medium is capable of sustaining aerobic metabolism for a period of at least 10 minutes, preferably at least about 25 minutes, without producing detectable levels of lactate or substantially depleting the metabolic substrates of the cell. After preconditioning, the same cell media is used as an effective medium for cryopreservation of the cells. Furthermore, the cell medium also can be used after storage during reconditioning and reoxygenation of the preserved cells.
The cell medium contains adenosine, typically in a concentration ranging from about 2.7 mM to about 3.6 mM. Preferably, the adenosine concentration ranges from about 2.9 mM to about 3.1 mM, and, more preferably, is about 3.0 mM. Adenosine is rapidly converted by cells into adenosine triphosphate. The adenosine triphosphate is a cell energy source immediately available to promote cell growth and regeneration during preconditioning.
The cell medium can also include other cell energy sources, such as saccharides. Any saccharide, such as glucose, or metabolite of the glycolytic pathway, such a pyruvate, a metabolite of glucose, can be added to the cell cryopreservation medium as a potential energy source. The cell medium of one embodiment contains glucose in an amount ranging from about 18 mM to about 24 mM. Preferably, the solution contains glucose in an amount ranging from about 19 mM to about 21 mM, and most preferably about 20 mM. The cell medium of another embodiment contains sodium pyruvate in an amount ranging from about 0.9 mM to about 1.1 mM, preferably, about 1.0 mM.
The cell medium also includes a calcium channel blocker to prevent the calcium-regulated export of adenosine from the cell. This allows higher levels of adenosine in the cell which fortifies the cell with rapidly available energy. An example of a suitable calcium channel blocker is verapamil. In one embodiment, verapamil is added to the cell medium in an amount ranging from about 0.04 mM to about 0.07 mM. It is added preferably in an amount ranging from about 0.05 mM to about 0.06 and more preferably in an amount of about 0.05 mM.
Importantly, the cell medium contains various cell metabolites, such as nutrients that are easily absorbed into the cells during preconditioning. For example, the cell medium preferably contains one or more amino acids. Since each of the amino acids can be absorbed by the cell and consumed during the cell""s normal metabolism, it is preferable to have a concentration of all amino acids in the cell medium. However, it is believed that the addition of any amino acid will serve to strengthen the cell during preconditioning, even if other amino acids are not present.
The concentration of the amino acids is chosen to match the amino acid concentration found in the healthy cytoplasm of the cells to be preconditioned. Alternatively, the concentration of amino acids in the medium is chosen to be proportional to the metabolic needs of the cells during normal cell metabolism. Some amino acids are used more frequently and should be present in higher amounts.
L-alanine is preferably added in an amount ranging from about 0.09 mM to about 0.11 mM and more preferably added in the amount of about 0.10 mM. Arginine is preferably added in an amount ranging from 0.6 mM to about 0.8 mM and is more preferably added in an amount of about 0.7 mM. Asparagine and/or aspartic acid are preferably added in a total amount ranging from about 0.9 mM to about 1.1 mM and most preferably added in an amount of about 1.0 mM. In one embodiment, the ratio of asparagine to aspartic acid is 10:1.
Cysteine is preferably added in an amount ranging from about 0.9 mM to about 1.1 mM and most preferably added in an amount of about 1.0 mM. Glutamine and/or glutamic acid are preferably added in a total amount ranging from about 2.7 mM to about 3.3 mM and more preferably added in an amount of about 3.0 mM. The ratio of glutamine to glutamic acid is about 2:1 in one embodiment of the present invention. Glycine is preferably added in an amount ranging from about 0.9 mM to about 1.1 mM and more preferably added in an amount of about 1.0 mM. Histidine is preferably added in an amount ranging from about 0.25 mM to about 0.30 mM and more preferably added in an amount of about 0.28 mM. Isoleucine is added in an amount preferably ranging from about 0.7 mM to about 0.9 mM and more preferably added in an amount of about 0.8 mM. Leucine is added in an amount ranging from 0.7 mM to about 0.9 mM and more preferably added in an amount of about 0.8 mM.
Lysine is added in an amount ranging from about 0.7 mM to about 0.9 mM and more preferably added in an amount of about 0.8 mM. Methionine is added in an amount ranging from 0.18 mM to 0.22 mM and more preferably added in an amount of about 0.20 mM. Phenylalanine is added in an amount ranging from about 0.35 mM to 0.45 mM and more preferably added in an amount of about 0.40 mM. Proline is added in an amount ranging from about 0.09 mM to about 0.11 mM and more preferably added in an amount of about 0.10 mM. Serine is added in an amount ranging from about 0.35 mM to about 0.45 mM and more preferably added in an amount of about 0.40 mM. Threonine is added in an amount from about 0.35 mM to about 0.45 mM and more preferably added in an amount of about 0.40 mM. Tryptophan is added in an amount ranging from 0.12 mM to about 0.15 mM and more preferably added in an amount of about 0.13 mM. Tyrosine is added in amount ranging from about 0.55 mM to about 0.65 mM and more preferably added in an amount of about 0.60 mM. Valine is added from about 0.7 mM to about 0.9 mM and more preferably 0.8 mM.
Additionally, the cell media preferably contains one or more vitamins selected from the group comprising pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin, and thiamine. Vitamins are supplied to fortify the cell after cell harvesting. They serve to replenish vitamins lost during treatment with the hydrolytic enzyme or that might have been deficient in the donor source of the cells.
While it is preferred that all of the known vitamins are added to the cell medium. the addition of vitamins are optional. In one embodiment, the following vitamins are added in the stated amounts. Pantothenate is added in an amount ranging from about 0.003 to about 0.009 mM and more preferably about 0.008 mM. Choline chloride is preferably added in an amount ranging from about 0.026 mM to 0.032 mM and more preferably about 0.029 mM. Folic acid is added in an amount ranging from about 0.008 mM to 0.010 mM and more preferably about 0.009 mM. Inositol is added in an amount ranging from about 0.036 mM to 0.044 mM and more preferably about 0.040 mM. Niacinamide is added preferably in an amount ranging from about 0.030 mM to 0.036 mM and more preferably about 0.033 mM. Pyridoxal is added in an amount ranging from about 0.018 mM to 0.022 mM and more preferably about 0.020 mM. Riboflavin is added in an amount ranging from about 0.0009 mM to 0.0011 mM and more preferably about 0.001 mM. Thiamine is added in an amount ranging from about 0.011 mM to 0.013 mM and more preferably about 0.012 mM.
In some embodiments, the cell medium contains one or more inorganic salt. The salt is added to the medium to prevent the cell reserves from being depleted. Since the inorganic ions forming the salts are very small, they readily pass through the cell membrane and are lost from the cell. Suitable inorganic anions include chloride, phosphate, sulfate, or selenite ions. Suitable inorganic cations include sodium, potassium, magnesium, copper, or zinc ions. In these embodiments, the cell medium preferably has a concentration of inorganic ions substantially equal to the intercellular concentration of the ions in a cell in vivo.
In preferred embodiments, the cell medium contains inorganic salts in an amount sufficient to create a slightly hypertonic medium, i.e., is slightly hypertonic compared to the osmolarity of the cell cytoplasm. The cells shrink slightly when in such a slightly hypertonic medium. Thus, there is less stress on the membrane which benefits the cell during cold preservation and cryopreservation. Accordingly, in a preferred embodiment, the cell medium has an osmolarity of approximately 330 mOsml/L.
The following concentration of salts have been found effective in maintaining appropriate ionic levels. Sodium chloride is added preferably in an amount ranging from 110 mM to about 120 mM and more preferably in an amount of about 115 mM. Potassium chloride is added preferably in an amount ranging from approximately 4.75 mM to 5.25 mM and more preferably in an amount of about 5 mM. Potassium phosphate is added preferably in an amount ranging from about 2.85 mM to about 3.15 mM and more preferably in an amount of about 3 mM. Magnesium sulphate is added preferably in an amount ranging from 1.15 mM to about 1.25 mM and more preferably in an amount of approximately 1.2 mM.
Copper sulfate is available in a hydrated form (CuO4.H2O) and is added in an amount ranging from approximately 8.5 mM to approximately 9.4 mM and more preferably 8.9 mM. Zinc sulphate is available in a hydrated form having a formula (ZnSO4.H2O) and is added in an amount ranging from 4.2xc3x9710xe2x88x928 mM to about 4.6xc3x9710xe2x88x928 mM and more preferably 4.4xc3x9710xe2x88x928 mM. Sodium selenite (Na2SeO3) is preferably added in an amount ranging from about 2.8xc3x9710xe2x88x926 through 3.2xc3x9710xe2x88x926 mM and more preferably 3.0xc3x9710xe2x88x926 mM.
Also in some embodiments, the cell cryopreservation medium contains at least one hormone. Suitable hormones include insulin, and preferably bovine pancreas insulin; dexamethasone; leutropic hormone and preferably sheep leutropic hormone; transferrin and preferably human transferrin; somatropin; linoleic acid; and fetal bovine serum.
Because the cell suspension is to be frozen, a cryopreservative is preferably added to the cell medium. Suitable cryopreservatives include DMSO, glycerol, propylene glycol, and other large molecules with a high bonding affinity to water, with DMSO being most preferred. Since DMSO is potentially toxic, it is not added to the cell medium until after the cell medium has been combined with the cells and the resulting suspension cooled to a temperature below that which DMSO adversely affects the cells. It is preferred that DMSO be added to the suspension at a temperature at or below about 4xc2x0 C. In one embodiment of the invention, the DMSO has a concentration ranging from about 8 vol. % to about 15 vol %. Preferably, the concentration of DMSO is about 12 vol. %.
It is also preferable for the cell medium to include an oxygen free-radical scavenger to protect the cells from any oxygen free-radicals that may be present during reoxygenation after storage. Oxygen free-radicals are highly reactive and cause considerable damage to a cell. Typically, oxygen free-radical scavengers are large organometallic molecules that readily react with the oxygen free-radicals, before the radicals can damage the cell.
Preferred oxygen free-radical scavengers include allopurinol and/or glutathione. In one embodiment, allopurinol is added in an amount ranging from about 0.9 mM to about 1.2 mM and is added more preferably in an amount of about 1 mM. In another embodiment, glutathione is added in an amount ranging from about 2.7 mM to about 3.6 mM. It is added preferably in an amount ranging from about 2.9 mM to about 3.1 mM and is added more preferably in an amount of about 3.0 mM.
Alternatively, the cell produces glutathione naturally when glutathione levels are low. Glutathione is formed from three amino acids, namely glycine, glutamate, and cysteine. Thus, the oxygen free radical scavenger function can be satisfied or, at least, supported by a medium that combines these three amino acids.
The cell medium of one embodiment is buffered. The normal pH of a cell is typically about 7.4 but can fluctuate under normal situations. In a healthy cell in vivo the pH will fall within a first range of from about 7.3 to about 7.5 without dramatically affecting the performance of the cell. During cell cooling, the pH of the cell becomes more acidic. As the cell becomes acidic, the enzymes of the cell do not function normally which arrests the cell metabolism and other enzymatic activity.
Conventional cell cryopreservation media are buffered so as to maintain the pH of the medium at the pH of natural extracellular fluid during the entire preconditioning and cryopreservation process. In contrast to such conventional media, in some embodiments, the buffer included with the inventive cell medium allows the pH of the medium to drop from about 6.3 to about 6.6, preferably form about 6.4 to about 6.5, and most preferably about 6.4, when the cell suspension is cooled to temperature of about 4xc2x0 C.
Preferred buffers include a sodium carbonate buffer, an N-[Hydroxyethyl]piperazine-Nxe2x80x2[2-ethananesulfonic acid] (xe2x80x9cHEPESxe2x80x9d) buffer or a combination of the two. In accordance with a most preferred embodiment, the amount of sodium bicarbonate ranges from about 22.5 mM to about 27.5 mM, preferably from about 24 mM to about 26 mM and, more preferably, about 25 mM, while the amount of HEPES ranges from about 9 mM to about 11 mM and more preferably about 10 mM.
When, the cell medium is combined with the cell samples at 37xc2x0 C., the pH of the mixture should be within the above stated first range of from about 7.3 to about 7.5 and should preferably be as close to 7.4 as possible. Then, as the sample is cooled, the pH will drop. When the pH is 4xc2x0 C., the pH should fall within the above stated second range of from about 6.3 to about 6.6, preferably form about 6.4 to about 6.5, and most preferably about 6.4. If the pH does not fall within the second range or it is desired that the medium have a pH closer to 6.4, the medium can be titrated with a mild sodium carbonate solution or a mild carbonic acid solution to adjust the pH to the desired value. One benefit of the inventive medium is that the pH will return to a pH close to 7.4 when the cells are warmed to 37xc2x0 C.
The method of using the cell medium is described in copending application by the inventors of the present patent application and which is filed the same day as the present application. This application is Ser. No. 09/168,366, now U.S. Pat. No. 6,140,123 and is fully incorporated by reference into the present application.
The method begins by taking harvested eukaryotic or prokaryotic cells and suspending the cells in the cell medium, while maintaining the temperature at about 35-40xc2x0 C., and more preferably about 37xc2x0 C. The medium is oxygenated by bubbling oxygen through the solution using a gas having an oxygen content of no less than 80 vol. %. In one embodiment, the medium is saturated at a pressure exceeding atmospheric pressure. The cells are preconditioned in this matter for a period ranging from about 10 minutes to about 48 hours. Preferably, the cells are preconditioned for a period ranging from about 10 minutes to about 2 hours, and more preferably about 25 minutes.
The cell suspension is then cooled to about 4xc2x0 C. DMSO is slowly added to the suspension until the amount of DMSO reached has a concentration ranging from about 8 vol. % to about 15 vol %., preferably about 12 vol. %. The suspension is placed in cryo-vials for freezing. Cryo-vials containing the suspension are first placed in a freezer that maintained at xe2x88x9270xc2x0 C. for about two hours to allow the water inside the cell to penetrate into extracellular space. Then the cryo-vials are transferred to the vapor phase of a liquid nitrogen tank for 10-24 hours. Thereafter, the cryo-vials are immersed in liquid nitrogen for long term storage at xe2x88x92196xc2x0, the boiling point of liquid nitrogen.
The cells are recovered by placing the frozen suspension in a water bath maintained at 37xc2x0 C.-42xc2x0 C. and gently agitating until the temperature of the cell suspension reaches 37xc2x0 C. The cell suspension is then transferred into a round bottom glass tube, spun to separate the cells from a supernatant, and the supernatant removed. Next the cells are washed with additional cell cryopreservation medium, including DMSO, and having an osmolarity of 1500 mOsml/l . The cell medium is slowly added to the cells and the cells resuspended. After resuspension, the cells are spun the supernatant again removed, and the cells recovered. The cells are then washed with an oxygenated cell cryopreservation medium that includes DMSO but has an osmolarity of 900 mOsml/l. Finally, the cells are resuspended in a cell cryopreservation medium having no DMSO, spun, and recovered. The cells are then ready for use.
The present invention also comprises a kit for storage of cells. The kit includes a cell medium as described above with a vessel for containing the cells. Also included in the kit is a set of instructions. The instructions include directions on using the cell medium in accordance with the principles of the present invention. In one embodiment, the instructions direct the user to oxygenate the cell medium and precondition the cells at a temperature of approximately 37xc2x0 C. for a length of time ranging from about 10 minutes to about 48 hours. According to this embodiment, the kit preferably includes a source of oxygen and an oxygen delivery device. The oxygen delivery device enables the medium to be saturated with oxygen.
The instructions according to another embodiment of the invention, provide directions to measure the pH of the medium when the medium is at a temperature of about 4xc2x0 C. The directions further include instructions on titrating the medium to a pH of about 6.3 to about 6.5, preferably about 6.4.
The present invention also includes a preserved cell sample that includes a sample of eukaryotic or prokaryotic cells suspended in a cell medium according to the principles of the present invention. The cell sample is preferably kept at a temperature below 4xc2x0 C. In another embodiment the cell sample is frozen and is preferably frozen at the boiling point of liquid nitrogen.
The following examples are included to further illustrate the invention. They are not limitations thereon.